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Due to global warming, animals are experiencing heat stress (HS), affecting many organic functions and species’ survival. In this line, some characteristics of immune cells in sea urchins subjected to short-term HS were evaluated. Paracentrotus lividus adult females were randomly divided into three groups and housed in tanks at 17 °C. In two of these tanks, the temperatures were gradually increased up to 23 and 28 °C. Celomatic fluid was collected after 3 and 7 days. The coelomocytes were morphologically typed and evaluated for their mitochondrial membrane potential (MMP), lipoperoxidation extent (LPO), and hydrogen peroxide content (H2O2). Respiratory burst was induced by treatment with phorbol 12-myristate 13-acetate (PMA). HS caused a significant change in the coelomocytes’ type distribution. MMP increased in the 23 °C-group and decreased in the 28 °C-group at both 3 and 7 days. LPO only increased in the 28 °C-group at 7 days. H2O2 progressively decreased together with the temperature increase. Respiratory burst was detected in all groups, but it was higher in the 17 °C group. In conclusion, the increase in temperature above the comfort zone for this animal species affects their immune cells with possible impairment of their functions.

Simple and inexpensive analytical methods for assessing redox balance in biological matrixes are widely used in animal and human diagnostics. Two of them, reactive oxygen metabolites (ROMs) and total oxidant status (TOS), evaluate the lipid hydroperoxide (LOOH) content of the sample and are based on iron-mediated mechanisms. However, these tests provide uncorrelated results. In this study, we compared these two tests in the blood serum of goat kids and lambs, together with an evaluation of ceruloplasmin (CP) oxidase activity. No significant correlation was found between ROMs and TOS, or between TOS and CP oxidase activity, in either species. Conversely, ROMs and CP oxidase activity were highly correlated in both kid and lamb samples (p < 0.001). A significant progressive reduction in the analytical signal in the ROMs assay was observed when sodium azide, an effective CP inhibitor, was added to the samples before the assay (p < 0.001). This decrease was related to sodium azide concentration (p < 0.01) and was not found when sodium azide was added at the same concentrations in the TOS assay. These findings suggest that ROMs, unlike TOS, may be affected by CP, which interferes with LOOH detection in blood samples.

The etiopathogenesis of endometritis in mares and cows differs significantly; this could depend on a different sensitivity and reactivity of the uterus but also on endocrine and rearing factors and different stress sources. In both species, microorganisms and the immune system play a primary role in the generation of this pathology. Microbiological and cytological tests support clinical examination and significantly improve diagnostic accuracy. For both species, during the inflammation, immune cells invade the endometrium and release bioactive substances to contrast primary or secondary pathogen contamination. These molecules are traceable to cytokines, chemokines, and prostaglandins as well as reactive oxygen and nitrogen species (ROS and RNS), collectively known as RONS. The RONS-mediated oxidation causes morphological and functional alterations of macromolecules, such as proteins, lipids, and nucleic acids, with the consequent production of derivative compounds capable of playing harmful effects. These bioactive molecules and by-products, which have recently become increasingly popular as diagnostic biomarkers, enter the bloodstream, influencing the functionality of organs and tissues. This review has collected and compared information obtained in cows and mares related to the diagnostic potential of these biomarkers that are assessed by using different methods in samples from either blood plasma or uterine fluid.

By exploiting the PMN property to produce high quantities of oxygen peroxide to neutralize pathogens, the oxygen peroxide content of uterine cells was measured to diagnose endometritis. After preliminary in vitro studies in which endometrial cells from slaughtered mares were mixed with leukocytes from peripheral blood, endometrial samples were collected by uterine flushing from mares before insemination. Staining endometrial cells with H2DCF-DA was combined with hydroethidine to normalize the fluorescence intensity with the cellular content of the sample. Stained cell smears were assumed as the gold standard of endometritis, and based on this assay, the samples were considered positive (C+) and negative (C−) for endometritis. The amount and the turbidity of fluid recovered by uterine flushing were significantly (p < 0.01) higher in C+ than in C−. Moreover, the oxygen peroxide content of the endometrial cells was significantly higher in the C+ than in the C− group (6.31 ± 1.92 vs. 3.12 ± 1.26, p = 0.001). Using the value of 4.4 as the cutoff level of this fluorescence cytology assay, it was found that only one C− sample exceeded the cutoff level (false positives = 7.7%) while three C+ samples showed values below the cutoff level (false negative = 11.5%).

Serological analysis may provide relevant information on endometritis diagnostics. Therefore, mares scheduled for AI with refrigerated semen, at the time of heat signs, underwent blood and uterine fluid samplings using a swab, uterine lavage for culture analysis, and treatment with human chorionic gonadotropin to induce ovulation. After 24–28 h, the mares were inseminated and, if positive at the culture test, treated with antibiotics chosen based on the susceptibility test. Uterine cells obtained by swabs were used for cytological examination with both classical and fluorescence techniques. Blood serum and uterine fluid samples were analyzed for assessing parameters related to redox balance, inflammation, and protease regulator potential. In blood serum, total antioxidant capacity, measured as the ferric reducing ability of plasma (FRAP), was significantly lower in cytologically endometritis-positive than -negative mares. In the uterine fluid, total thiol levels (TTL), nitric oxide metabolites (NOx), protease activity and total protein content varied significantly between groups. Although the cytological examination was more capable of discriminating between endometritis-positive and -negative mares in relation to the parameters examined, no statistically significant differences emerged in terms of pregnancy rate in relation to cytological and culture diagnosis as well as in mares diagnosed as positive and negative for endometritis.

Seminal plasma (SP) of donkey stallions was evaluated using various oxidative stress parameters as well as protease and protease inhibitor activities. SP was obtained by nine donkey stallions. In addition, one donkey stallion with non-obstructive azoospermia was enrolled in this study. Free radical scavenging activity (FRSA), the ferric reducing ability of plasma (FRAP), total antioxidant capacity (TAC), and total thiol level (TTL) were highly correlated with each other and with the protease inhibitor activity. However, only FRAP, TAC, and the nitrate/nitrite concentration (NOx) were significantly correlated with sperm concentration, production, and kinetics. Protease inhibitor activity was highly correlated with sperm concentration and production; however, it did not correlate with sperm kinetics. The azoospermic stallion produced a lower amount of semen than the normospermic stallions and its SP showed a lower antioxidant activity when evaluated with FRAP, TAC, and TTL as well as a higher NOx and a lower protease inhibitor activity. In conclusion, the evaluation of SP oxidative profile by FRAP, TAC, and NOx may provide reliable information on donkey sperm quality whereas protease inhibitor activity may play a role as a marker of the sperm concentration in this species.

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